Systems and methods for producing metal clusters; functionalized surfaces; and droplets including solvated metal ions

ABSTRACT

The invention generally relates to systems and methods for producing metal clusters; functionalized surfaces; and droplets including solvated metal ions. In certain aspects, the invention provides methods that involve providing a metal and a solvent. The methods additionally involve applying voltage to the solvated metal to thereby produce solvent droplets including ions of the metal containing compound, and directing the solvent droplets including the metal ions to a target. In certain embodiments, once at the target, the metal ions can react directly or catalyze reactions.

RELATED APPLICATIONS

The present application claims the benefit of and priority to each ofU.S. provisional application Ser. No. 61/877,528, filed Sep. 13, 2013,U.S. provisional application Ser. No. 61/880,219, filed Sep. 20, 2013,and U.S. provisional application Ser. No. 62/012,619, filed Jun. 16,2014, the content of each of which is incorporated by reference hereinin its entirety.

GOVERNMENT SUPPORT

This invention was made with government support under DE-FG02-06ER15807awarded by the U.S. Department of Energy. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention generally relates to systems and methods for producingmetal clusters; functionalized surfaces; and droplets including solvatedmetal ions.

BACKGROUND

In recent years, metallic nanoparticles and metal clusters have beenembraced by industrial sectors due to their applications in the field ofelectronic storage systems, optics and medical imaging, biotechnology,magnetic separation and pre-concentration of target analytes, targeteddrug delivery, and vehicles for gene and drug delivery. Generally, ananoparticle (or nanopowder or nanocluster or nanocrystal) is a smallobject that behaves as a whole unit in terms of its transport andproperties. Typically, such particles have at least one dimension lessthan 100 nm. Generally, a cluster is an ensemble of bound atomsintermediate in size between a molecule and a bulk solid. A metalcluster contains a group of two or more metal atoms in which direct andsubstantial metal-metal bonding is present.

The adoption of this technology is hindered by the difficultiesassociated with producing metallic nanoparticles and metal clusters. Forexample, there is no existing process that allows for the directproduction of metallic nanoparticles from a starting metal. The typicalproduction process employs a strong acid to oxidize the starting metalto produce a precursor metallic salt. That metallic salt is thenpurified and isolated. The purified and isolated metallic salt is thenchemically reduced using reducing agents (such as NaBH₄) in bulksolution in the presence of capping ligands to produce the metalnanoparticles. The existing process is complex, requiring multipleseparate reactions, in separate reaction steps, and is costly.

Metal clusters are traditionally produced in vacuum using laserablation. A laser is applied to a metal target to vaporize the metalunder vacuum, thereby producing metal cluster ions. The metal clusterions are then contacted to a chemical reducing agent to reduce the metalcluster ions to neutral metal clusters. The existing process is costly,requiring expensive lasers and vacuum chambers, and also requireschemical reducing agents. Those processes that do operate at atmosphericpressure are difficult to control.

SUMMARY

In certain aspects, the invention generally relates to systems andmethods for producing droplets including solvated metal ions. The metalcan be separated from the droplet as a nanoparticle and can be generatedin situ and react in situ. This aspect of invention provides systems andmethods for producing metallic nanoparticles directly for a startingmetal. Aspects of the invention are accomplished using solvent andligand aided spray ionization of metals. Metals are directly ionizedusing appropriate solvents to produce droplets that include solvatedmetal ions. The droplets including the solvated metal ions are directedto a reactive surface where the solvated metal ions are reduced to formthe metallic nanoparticles. In that manner, systems and methods of theinvention, eliminate the need for metallic salt precursors, dramaticallysimplifying the process of producing metallic nanoparticles.

In certain embodiments, the invention provides methods for providingdroplets including solvated metal ions. The methods may involvecontacting a metal (e.g., a noble metal) metal-containing compound witha solvent and applying voltage to produce solvent droplets that includethe solvated metal ions. The solvent droplets including the solvatedmetal ions are then directed to a target. The solvent droplets may bedirected to the target by an electric field, such as the field generatedby the voltage source that applies voltage to the metal containingcompound. The solvent droplets may also be directed to the target by agas flow. In certain embodiments, both an electric field and a gas floware used to direct the solvent droplets to the target.

Generally, the target is a reactive surface. The invention encompassesnumerous types of reactive surfaces. For example the reactive surfacemay be a surface that includes a cathode, with or without a cathodesolvent. In such embodiments, interaction of the solvated metal ionswith the cathode reduces the solvated metal ions to a neutral state. Inother embodiments, the reactive surface is a surface that includes areducing reagent.

In certain embodiments, interaction of the solvated metal ions in thedroplets with the reactive surface generates at least one metalcontaining product, such as a metallic nanoparticle. In certainembodiments, methods of the invention further involve contacting acapping ligand to the metal ions on the reactive surface to stabilizeand protect the nanoparticle.

While exemplified for production of metallic nanoparticles, aspects ofthe invention have other applications. Accordingly, while the target isa reactive surface is certain embodiments, the target is not limited tobeing a reactive surface. For example, the target may be an analyticalinstrument, such as a mass spectrometer. In those embodiments, thesolvent droplets including the solvated metal ions are directed into theanalytical instrument for analysis. After analysis the metal ions maystill be collected, for example on a surface. These embodiments alsoallow selection of a subset of metal ions for collection. In otherembodiments, the target is a reaction mixture. In those embodiments, thedroplets including the solvated metal ions are directed into thereaction mixture. In certain embodiments, contact of the solvated metalions to the reaction mixture catalyzes a reaction in the reactionmixture.

Other embodiments of the invention provide methods for producing metalcontaining nanoparticles. Those methods involve providing a metalcontaining anode in contact with a solvent, applying voltage to theanode, thereby producing solvent droplets comprising solvated metalions, directing the solvent droplets comprising the solvated metal ionsto a reactive surface, in which interaction of the metal ions with thereactive surface produces metal containing nanoparticles.

Still other embodiments of the invention provide systems for producingmetal containing nanoparticles. Those systems may include a dropletemitter including a metal containing anode and a solvent vessel operablycoupled to the anode, a high voltage source coupled to the dropletemitter, and a reactive surface positioned to receive droplets includingsolvated metal ions produced by the droplet emitter. The reactivesurface is functionalized to reduce solvated metal ions that interactwith the surface to thereby produce metal containing nanoparticles onthe surface. Systems of the invention may further include the solventwithin the solvent vessel. In certain embodiments, the system furtherincludes a gas flow generating device that is operably coupled to thedroplet emitter. In certain embodiments, the reactive surface includes acathode. In other embodiments, the reactive surface includes a reducingagent. In certain embodiments, the reactive surface includes both acathode and a reducing agent.

In other aspects, the invention generally relates to systems and methodsfor producing metal cluster ions at atmospheric pressure, therebyallowing neutral clusters to be generated. The invention providessystems and methods for direct and controllable synthesis of metalclusters at atmospheric pressure without the need for chemical reducingagents. The methods of the invention allow for cluster sizes to bemeasured and conditions to be adjusted in order to change the size ofthe produced metal cluster. Such aspects of the invention areaccomplished using spray ionization of metal salts. First, a solutionphase of metal salt clusters is sprayed, generating neutral as well ascharged clusters. The solvent is evaporated to give a charged saltcluster which is heated to remove the anionic portion leaving a nakedmetal cluster cation. The metal cluster ions may be, although notrequired, directed to a surface where the metal cluster ions are reducedto form the metal clusters, which may occur at atmospheric pressure orunder vacuum. In that manner, systems and methods of the inventioneliminate the need for chemical reducing agents, lasers and vacuums,thereby dramatically simplifying the process of producing metal clustersand allowing them to be utilized in air.

In certain embodiments, the invention provides methods for producingmetal cluster ions at atmospheric pressure. Methods of the inventioninvolve applying voltage and heat to a metal salt (such as a noble metalsalt) at atmospheric pressure to thereby ionize the metal salt andproduce metal cluster ions. The metal cluster ions are then directed toa target. The metal cluster ions may be directed to the target by anelectric field, such as the field generated by the voltage source thatapplies voltage to the metal salt. The metal cluster ions may also bedirected to the target by a gas flow. In certain embodiments, both anelectric field and a gas flow are used to direct the metal cluster ionsto the target. A heat source is used to heat the metal salt. The heatsource may be a heat coil or other type of heat source, such as a heatedchamber. Alternatively, a directed heat source, such as a heat gun thatcan generate and directed a heated gas flow, may be used to heat themetal salt. In certain embodiments, the metal salt is taken up in asolvent as the first step in metal cluster formation. In certainembodiments, the metal cluster ions react with the solvent.

The target may be at atmospheric pressure or under vacuum. Generally,the target is a surface, and in certain embodiments, the surface isfunctionalized to be a reactive surface. The invention encompassesnumerous types of reactive surfaces. For example the reactive surface isa surface that includes a reducing reagent. In such embodiments,interaction of the metal cluster ions with the reactive surface reducesthe metal cluster ions to a neutral state. The metal clusters can benaked or unprotected metal clusters. Alternatively, the metal clustersmay be protected metal clusters. In embodiments that produce a protectedmetal clusters, methods of the invention further involve contacting acapping ligand to the metal cluster ions on the surface to stabilize andprotect the metal cluster.

While exemplified for production of metal clusters, aspects of theinvention have other applications. Accordingly, while the target is asurface is certain embodiments, the target is not limited to being asurface. For example, the target may be an analytical instrument, suchas a mass spectrometer. In those embodiments, the metal cluster ions aredirected into the analytical instrument for analysis. After analysis themetal cluster ions may still be collected, for example on a surface.These embodiments also allow selection of a subset of metal cluster ionsfor collection. An exemplary system uses quadrupole filters to selectwide mass ranges appropriate to individual sizes of clusters ofinterest. In certain embodiments, the analytical instrument is used tooptimize conditions that favor certain metal cluster sizes or ranges ofsizes.

In other embodiments, the target is a reaction mixture. In thoseembodiments, the metal cluster ions are directed into the reactionmixture, which may be in an ambient environment. In certain embodiments,contact of the metal cluster ions to the reaction mixture catalyzes areaction in the reaction mixture.

Another embodiment of the invention provides systems for producing metalclusters. Systems of the invention include a droplet emitter atatmospheric pressure, a high voltage source coupled to the dropletemitter, a heating element operably coupled to the droplet emitter, anda surface positioned to receive metal cluster ions produced by thedroplet emitter in which deposition of the metal cluster ions of thesurface produces metal clusters. In certain embodiments, the surface isat atmospheric pressure. In other embodiments, the surface is undervacuum. In certain embodiments, the system further includes a solventvessel containing a solvent in which the solvent vessel is operablycoupled to the droplet emitter. In certain embodiments, the systemfurther includes a gas flow generating device that is operably coupledto the droplet emitter. In certain embodiments, the system furtherincludes a mass analyzer positioned between the droplet emitter and thesurface. The mass analyzer allows for spatial selective soft landing ofthe metal cluster ions onto the surface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing illustrating a prior art method for producing goldnanoparticles.

FIG. 2 is a drawing illustrating methods of the invention for producinggold nanoparticles.

FIG. 3 is a drawing illustrating an embodiment of a system of theinvention.

FIGS. 4A-4B are transmission electron microscopy images of prepared gold(FIG. 4A) and silver (FIG. 4B) nanoparticles in less than 5 minutes.(The scale bar is 50 nm.) The sizes, shapes and dispersity of thenanoparticles can be controlled by altering the nature of the ligandsand the experimental conditions.

FIG. 5 is a drawing illustrating the reduction of 4-nitrophenol by NaBH₄using gold nanoparticles as catalyst. This is reaction is widely used toprobe the surface reactivity of synthesized nanoparticles.

FIG. 6 panels A-D show that prepared (both offline and in-situ)nanoparticles can be used to catalyze chemical reactions. FIG. 6 panelsA-D are graphs showing overlays of UV-vis spectra of the nitrophenolreaction mixture monitored every 30 seconds. The major peak at 400 nmcorresponds to the n→π* transition of 4-nitrophenol anion and itsdecrease in (panel B) and (panel D) represents the progress of thereduction. (Panel A), Without AuNP, the reaction did not proceed at allin 12 hours. (Panel B) Adding off-line prepared electrosprayed AuNP intothe reaction system, the reaction was complete within 30 minutes. (PanelD) Online addition of Au⁺ ions, instead of AuNP, sprayed into thereaction mixture at the rate of 10 nA while UV-vis was monitored. Thisextremely small amount of gold catalyzed the reduction reaction. (PanelC) Control experiment that sprays protic solvents to the reactionmixture to prove that the reaction is not influenced by cathodicelectron currents.

FIG. 7 is a transmission electron microscope (TEM) image of ananoparticle composed of a silver core surrounded by and attached tosmaller gold spheres.

FIG. 8 is a TEM image of a nanoparticle composed of a gold coresurrounded by silver sphere. Silver has lower electron density resultsin low color tone in the TEM figure.

FIG. 9 is a schematic show direct generation of cuprous organometallicfrom its metal form, note the structure on the right is the catalyst foratom transfer radical polymerization.

FIG. 10 is graph showing analytical results of synthesized polystyreneusing gel permeation chromatography (GPC) and generated by nanoparticlecatalyzed reaction. The average mass is 14 k Da.

FIG. 11 is a schematic showing a prior art method of producing metalcluster ions.

FIG. 12 is a schematic showing another prior art method of producingmetal cluster ions.

FIG. 13 is a schematic showing methods of the invention for producingmetal cluster ions.

FIG. 14A is an ion chronogram of Ag₃ ⁺ (appearing as a set of isotopicpeaks in the region m/z 320-328) as a function of temperature. FIG. 14Bis a representative mass spectrum of 1 mM silver acetate that has beenpassed through a heated coiled loop at 215° C. The [Ag₃]⁺ represents 17%of the total ion current.

FIG. 15 panel A is a mass spectrum showing atmospheric ion/moleculereaction of silver clusters with isopropyl alcohol. Ligation of 1 and 2isopropyl alcohol were observed with the silver trimer. FIG. 15 panel Bshows MS² of m/z 384 confirming the ligation of 1 isopropyl alcohol.FIG. 15 panel C shows MS² of m/z 443 and FIG. 15 panel D shows MS³ ofm/z 384 confirming the ligation of two metal ligands.

FIG. 16 panel A shows two AgNP containing spots were created on top of apenny coin by metal electrolytic spray ionization deposition underambient conditions. The above spot was created before drop castingsample while the lower spot was created on top of a layer of sample(crystal violet). 10 ML of silver ions were landed to create the twospots. Similar morphology (FIG. 16 panels C and D) and enhanced Ramansignal (FIG. 16 panel B) were obtained from the two spots.

FIG. 17 panels A-D show morphologies of surface nanostructures createdby depositing different amount of silver ions onto a copper foil usingmetal electrolytic spray ionization deposition. Coverage turned out tobe one determining factor for the SERS performance of spots created bythis surface modification method.

FIG. 18 panel A is a dark field image of AgNP patterns created using agrounded TEM grid as mask, 90 μm squares separated by 10 μm columns.FIG. 18 panel B is a bright field image of AgNP patterns created using afloated stainless steel mesh, the pattern is composed of 20 μm diametercircles of AgNP. Black spots are dust from the ambient air. FIG. 18panel C shows that a similar AgNP pattern was created on top of copperfoil and each spot was SERS active. FIG. 18 panel D shows that whenimaged by Raman, signal of the dropcast (3 mm circle) crystal violetsample can only be observed in the AgNP regions. The scale bar in (d) is10 μm. Note that the nanoparticle spot (20 microns) is about 5 timessmaller than the aperture (90 microns) due to autofocussing by chargebuildup on the insulating aperture.

FIG. 19 panels A-C is a set of SERS spectra and average enhancementfactor of crystal violet (10⁵ per μm²) on top of AgNP nanostructure ontop of copper foil under excitation of (FIG. 19 panel A) 532 nm, 20 mW(FIG. 19 panel B) 633 nm, 8.6 mW (FIG. 19 panel C) 785 nm lasers, 52 mW.

FIG. 20 is a SERS spectra of R6G (10⁵ per μm²) on top of AgNPnanostructure on top of copper foil under excitation of 633 nm lasers.The two spectra were taken with (top) 1 second (bottom) 0.01 secondrecording time from the same spot region. The peak height (relative tobaseline) of 1365 cm⁻¹ band were labeled for spectra.

FIG. 21 shows Raman imaging of two spots created in an array. Crystalviolet (105 per μm²) was applied over the whole region by drop castingon top of copper foil. Raman signal of CV can only be observed whereAgNP spots were fabricated. The uniformity of the array is demonstratedby the similarity of the Raman images of CV from two randomly selectedspots in this array pattern. The volcano shape of the Raman imagingmight be due to the flux distribution of depositing ion plume whenfocused to 20 μm.

FIG. 22 panels A-D show AgNP structures created by 10 monolayer coverageof Ag⁺ deposited on top of Aluminum foil. Polydispersed morphology wasuniform throughout each spot created. Panels A-D are images from fourrandomly selected regions located >200 μm from each other in the samedeposition spot.

FIG. 23 shows different SERS signal intensity from surfaces modifiedwith different Ag coverage on a copper foil.

FIG. 24 panel A is a 4.9 mm² AgNP spot created on top of copper foil bydirect deposition without using any focusing or masking. This spot ofaveraged 8 ML coverage of silver was created by 136 min deposition of 13nA landing current. FIG. 24 panel B is an image showing that using aperforated plastic tape (50 μm thick, ˜500 μm diameter) on top of thedeposition target, only 17 min of deposition was needed to get twice asmuch coverage for this 0.23 mm² spot in less than 17 min, even thoughthe total depositing current dropped to 8 nA. Improved color uniformitywas also achieved by this spot compared to panel A. FIG. 24 panel C isan image showing that an array of even smaller spots was created byusing arrayed mask. FIG. 24 panel D is an image showing 100 μm spotsseparate by <2 mm.

FIG. 25 is an image showing an array of (20 μm) AgNP spots deposited ontop of a copper foil using a floated conductive stainless steel mesh asmask. The spots are 560 μm away from each other. The mask used is shownin the FIG. 26 and importantly it has much larger apertures than thespot sizes because of the focusing property of the electrically floatedmesh.

FIG. 26 is an image showing the stainless mesh used to create thepattern shown in FIG. 25. This mesh is a flat 200 μm thick platecomposed of with ˜200 μm holes. The floating/insulation from ground isachieved by 50 μm separation with the deposition target by plastic tape.

FIG. 27 is an image showing AgNP structures created by monolayercoverage of Ag⁺ deposited on top of gold foil. This surface has by farthe highest SERS performance. At the same time, the AgNP on this surfaceis also the one most susceptible to electron beam inducedmelting/aggregation during SEM image.

FIG. 28 panels A-B is an image showing AgNP structures created by on topof (panel A) ITO coated slide and (panel B) aluminum coated glass slide.

FIG. 29 panel A shows a cross section scan. FIG. 29 panel B is an imageshowing the reconstructed contour plot of the ion intensity at thedeposition surface as measured by the scan of the ionCCD. The elongationalong the x-axis of the figure is due to distortion of the IonCCD. FIG.29 panel C is an image showing a spot created by depositing silver ionfor 12 hours with an average coverage of 100 ML. FIG. 29 panel D is animage showing on the edge of the spot from FIG. 14 panel C, where theactual coverage varied due to the current density drop, a rainbow-likecolor transition was observed. This means that the surface plasmonresonance of this modified surface area can be tuned by just varying thecoverage of depositing silver ions between 0 and 100 ML.

FIG. 30 panels A-H are images showing particles and aggregates with arange of plasmon scatterings were generated by this one pot iondeposition method.

FIG. 31 shows different scattered spectra from the selected particlesand aggregates. With 10 ML coverage, the scattering spectra have widepeaks ranging between 510 nm and 650 nm, suggesting a broad range ofsurface plasmon resonance of surface prepared similarly. This“fuzziness” allowed for SERS activity for lasers with multiplewavelengths.

FIG. 32A shows that ions (279 m/z) originating at the nanoESI tipdiffuse outward and are subsequently focused to the deposition surfaceby the conductive mask through a 200 μm hole. Potentials applied to thenanoESI spray tip, and deposition surface were 1500V, 1000V, and 0Vrespectively. FIG. 32B is a close up view demonstrating ion (m/z 279)focusing occurring at the mask electrode. Ions were initialized 250 μmfrom the mask electrode. Spot size at the deposition surface isapproximately 20 μm. Focusing causes a 100× decrease in beam crosssection.

DETAILED DESCRIPTION

The invention generally provides systems and methods for the directsynthesis of metal containing products, such as a metallic nanoparticlesand/or metal clusters, from noble metals (such as gold) or noble metalsalts without using oxidizing and/or reducing reagents. In certainaspects, the invention generally relates to systems and methods forproducing droplets including solvated metal ions. In other aspects, theinvention generally relates to systems and methods for producing metalcluster ions at atmospheric pressure, thereby allowing neutral clustersto be generated. In other aspects, the invention generally relates tofunctionalized substrates, such as substrates functionalized for surfaceenhanced Raman Spectroscopy, methods of making thereof, and methods ofpatterning surfaces.

Systems and Methods for Producing Droplets Including Solvated Metal Ions

In certain embodiments, direct ionization of metals (including noblemetals) under ambient conditions is achieved using electrospray withorganic solvents. For example, gold is directly ionized to gold (I)cations in acetonitrile solution and the solvated metal cations are thencarried to a target by spraying micro-sized charged droplets that can bedirected by electric fields or by a gas stream. The target can be ananalytical instrument or it can be a collection medium where thedroplets are allowed to deposit and form a nanoparticle or to react forexample in a catalytic reaction presumably via formation of ananoparticle. The chemical environment of the deposition medium isindependently manipulated and this allows for the transformation ofmetallic ions species (e. g. gold(I)) into other forms of the element.Those products (metallic, organometallic, etc.) can be collected andthen used to perform further chemical manipulations, including variouscatalytic reactions.

A nanoparticles generally refers to a small object that behaves as awhole unit with respect to its transport and properties. Nanoparticlestypically have at least one dimension between 1 and 100 nanometers,although particles that are smaller or larger, e.g., 200 nanometers, 500nanometers, 1000 nanometer, or 2000 nanometers are also considerednanoparticles for purposes of the invention. Metallic nanoparticles arefurther described, for example in Mody et al. (J Pharm Bioallied Sci.,2(4): 282-289, 2010), the content of which is incorporated by referenceherein in its entirety.

FIG. 1 illustrates a prior art method of producing metallicnanoparticles. The most common methods of generation of nanoparticlesand metal clusters use chemical reduction of precursor compounds usingreagents (such as NaBH₄) in bulk solution in the presence of cappingligands. This allows reduction to the metal, clustering to formnanoparticle assemblies of the desired size and protection of theseassemblies. The precursor compounds are the corresponding metal salts(such as HAuCl₄) produced in separate steps involving oxidation ofmetals by acids and purification and isolation of the salts. (See FIG.1).

In contrast to the prior art method described in FIG. 1, systems andmethods of the invention allow for production of metallic nanoparticlesdirectly from a starting metal. That process is illustrated in anexemplary manner in FIG. 2, in which gold is used as the exemplarystarting metal. One of skill in the art will appreciate that the methoddescribed in FIG. 2 and throughout this application applies to any metalcontaining compound, such a noble metals or organometallic compounds,and is not limited to gold or indeed to metals. In the method shown inFIG. 2, the gold is contacted with a solvent and voltage is applied tothe gold and solvent producing an electrolysis spray. The spray issolvent droplets that include dissolved gold ions. Those dropletsincluding the metal ions are directed to a reactive surface, whereeither a cathode or a reducing agent is used to reduce the charged ionsto produce the gold nanoparticles. While FIG. 2 shows using either acathode or a reducing agent, in some embodiments, both a cathode and areducing agent are used. Directing ions to a surface and landing ions isdescribed, for example in International patent application numberPCT/US13/41348, and U.S. Pat. No. 7,361,311, the content of each ofwhich is incorporated by reference herein in its entirety.

FIG. 3 illustrates an embodiment of a system of the invention. Systemsof the invention include a droplet emitter. Such an emitter may be astandard electrospray probe, which is well known in the art. Such probesare described, for example in Fenn et al. (Science, 246(4926):64-71,1989), Ho et al. (Clin Biochem Rev, 24(1): 3-12, 2003), and Rehman (U.S.patent application number U.S. 2012/0012453) the content of each ofwhich is incorporated by reference herein in its entirety. The dropletemitter includes a metal anode coupled to a solvent vessel. The anode isconfigured such that a solvent in the vessel is able to interact withthe metal anode. The metal anode of the droplet emitter is coupled to avoltage source. Application of voltage to the metal in the solventcauses droplets of solvent including metal ions to be produced andemitted from the emitter. The solvent droplets including the metal ionsare then directed to a reactive surface.

The invention encompasses numerous types of reactive surfaces. Forexample the reactive surface may be a surface that includes a cathode,with or without a cathode solvent, as shown in FIG. 3. In suchembodiments, interaction of the metal ions in the solvent droplets withthe cathode reduces the metal ions to a neutral state. In otherembodiments, the reactive surface is a surface that includes a reducingreagent. Using systems of the invention, metallic nanoparticles of anytype may be produced, such as gold nanoparticles (FIG. 4A) or silvernanoparticles (FIG. 4B).

Different combinations of solvent and voltage allow the generation ofmetallic ion species with different oxidation states individually orcollectively, including those that are labile toward disproportionationwhen stored in bulk. The chemical environment of the deposition mediumis independently manipulated and this allows for the transformation ofmetallic ions species. The different chemical environment of the targetdeposition medium allows, for example, synthesis of nanoparticles andclusters; synthesis of organometallic compounds including catalysts; andmetal tagging by reaction with other neutral molecules. Table 1 belowshows different combination of solvents, metals, and reactive surfacesfor producing different types of metallic nanoparticles. Each column isindependent of each other and multiple combinations may be used.

TABLE 1 Anode Cathode Capping Reducing solvents Metal solvents ligandsreagent Pyridine Gold None None Cathodic electron Ammonia Silver WaterSodium NaBH₄ citrate Dimethyl Copper Toluene Tetraoctylammonium Metalion sulfoxide bromide itself Acetonitrile Platinum Ethanol Cetyl(disappropriation) trimethylammonium bromide Dimethylformamide PalladiumHeptane Dodecanthiol Dithiothreitol Ruthenium Chitosan Ethyl aldehyde

Systems and methods of the invention eliminate the use of strongoxidizing acids in producing metal precursor reagents from the metal,and eliminate the use of reducing agents in certain syntheses ofnanoparticles.

Without being limited by any particular theory or mechanism of action,it is believed that the metallic elements are introduced into thesolution in the form of weakly chelated cations (without counter ions orwith a deficiency of counter ions), as opposed to using metal salts inconventional methods. The polar nature of droplets containing the metalions allows a wider range of chemistry at the landing site includingeasier reduction of the metal.

The quantity of precursor generated and delivered to the target spot, iscontrolled by an electrical system, as opposed to weight/volume-basedmeasuring systems used in current solution phase methods. That allowsfor the synthesis of the final products in amounts ranging frominfinitesimal to bulk quantities. These features, combined with theadvantages discussed above, allow single-layer synthesis of metallicnanoparticles and other products on surfaces in an easily controllableway. The controllable small amount of evenly distributed ion depositionalso allows for easier synthesis of nano alloys. The spray can be usedto catalyze reactions e.g. polymerization in a solution.

Systems and methods of the invention allow for production of metallicnanoparticles in high yield with little waste. The whole process may bethought of as essentially a one-pot reaction with high atomicefficiency. Contact with conventional glassware is also eliminatedremoving a source of waste inevitable in conventional mass transferprocesses. The metal ions/clusters are generated as and when needed. Themetal ion sources can be easily and cheaply preserved and transported.

When used in surface patterning, this method has important advantages inthat it eliminates the bulk chemical reaction handling processes. Themetal ions and other reagents are contained in charged droplets guidedby electric fields or gas streams toward the target surface.

Systems and methods of the invention lower the cost and time in theproduction of derived products. The patterning of a single surface (3cm² using a single sprayer) with nanoparticles can be accomplished in aslittle as five minutes.

Droplet-transport of metal ions allows a wide range of chemicalreactions to be used. Because the solvents are unreactive, weakerligands can be used compared to the counter ions of corresponding metalsalts used in all current methodologies.

When used in catalysis, the nanoparticles can be prepared offline orthey can be prepared in situ by directly spraying the metal ioncontaining droplets into the target.

When preparing larger quantities of metal containing products, landingthe metal cation containing droplets to a target liquid medium isself-stirring. The mixing is achieved by numerous micro-dropletsdeposition events and a self-stirring phenomena, as opposed to themechanical stirring in the conventional methods. When droplet lands onthe liquid cathode surface, it merges but does not mix immediately. Theself-stirring phenomena is the result of the momentum from the impingingdroplets. Initial kinetic energy of the droplet (from the pneumaticforce of the sprayer) pushes the droplet moving forward for somedistance.

Systems and Methods for Producing Metal Cluster Ions

In certain embodiments, the invention generally provides systems andmethods for producing metal cluster ions at atmospheric pressure. Acluster is an ensemble of bound atoms intermediate in size between amolecule and a bulk solid. A metal cluster contains a group of two ormore metal atoms where direct and substantial metal-metal bonding ispresent. Typically, a cluster includes aggregates of 5 to 10⁵ atomic ormolecular units, but metal clusters of the invention are not limited tothis aggregate range and clusters of the invention may include a greateror lesser number of molecular units with very small clusters being veryeasy to create. Metal clusters may be homometallic, in which the samemetals are bound to each other. Alternatively, metal clusters may beheterometallic, in which different metals are bound to each other. Metalclusters of the invention may be noble metal clusters or other types ofmetal clusters, such as organometallic clusters, metal halide clusters,transition metal clusters, etc. Metal clusters of the invention may benaked (also known as unprotected) or protected, such as by a cappingligand. Metal clusters are further described, for example in Schmid etal. (Phil. Trans. R. Soc. A, 368(1915):1207-1210, 2010), the content ofwhich is incorporated by reference herein in its entirety.

Generally, naked or unprotected noble metal clusters are traditionallyproduced in vacuum using laser ablation. FIGS. 11-12 show prior artmethods of producing metal ion clusters. FIG. 11 shows a prior art laservaporization method. That method is performed entirely under vacuum. Inthat method, a laser is directed to impinge on a metal target, tothereby vaporize the metal into metal clusters in a plasma plume. Themetal clusters in the plume are condensed under vacuum to produce gasphase metal cluster ions that are under vacuum. The methods involvepulsed-laser vaporization, continuous-operating ovens, fast atombombardments, secondary ion sputtering, and arc discharge sources. FIG.12 shows a prior art ligand stripping method. In that method, preformedligated metal clusters are ionized and then transformed into naked metalclusters by ligand stripping in vacuum through collision induceddissociation.

Unlike prior art methods, the embodiments described herein generallyprovide systems and methods for producing metal cluster ions atatmospheric pressure. Systems and methods of the invention combineelectrospray ionization of appropriately chosen metal salts with modestheat to achieve similar results, at atmospheric pressure (FIG. 13). Forexample, using silver salts (e.g. silver acetate) the experimentalconditions can be adjusted to produce predominantly Ag₃ ⁺ or Ag₅ ⁺ ionsin the spray. The trimeric silver clusters interact in ambient air withvapors of alcohols and other reagents to give adducts such as [Ag₃ ⁺M₂].The metal clusters can be deposited on surfaces in air or in vacuum andused as reagents in heterogeneous reactions.

Systems of the invention include a droplet emitter. Such an emitter maybe a standard electrospray probe, which is well known in the art. Suchprobes are described, for example in Fenn et al. (Science,246(4926):64-71, 1989), Ho et al. (Clin Biochem Rev, 24(1): 3-12, 2003),and Rehman (U.S. patent application number U.S. 2012/0012453) thecontent of each of which is incorporated by reference herein in itsentirety. The droplet emitter is operably coupled to a heat source. Theheat source may be a chamber that heats a gas (e.g., air or nitrogen orother gas) surrounding the probe such that sprayed metal salts areheated to approximately 200° C. The heat source may also be a heat gun,or other directed heating source that can direct a flow of heated gasonto the metal salt. In certain embodiments, the heat source is a heatedcoil loop through which the metal salt passes. The metal salt isgenerally in contact with a solvent. In certain embodiments, the metalsalt reacts with the solvent. In other embodiments, the metal salt doesnot react with the solvent. Application of voltage and heat to the metalsalt produces metal cluster ions, as is shown in FIG. 13. The metalcluster ions are then directed to a target. Directing ions to a surfaceand landing ions is described, for example in International patentapplication number PCT/US13/41348, and U.S. Pat. No. 7,361,311, thecontent of each of which is incorporated by reference herein in itsentirety.

In certain embodiments, the target is a surface, such as a reactivesurface. The invention encompasses numerous types of reactive surfaces.For example the reactive surface may be a surface that includes acathode, with or without a cathode solvent. In such embodiments,interaction of the metal cluster ions with the cathode reduces the metalcluster ions to a neutral state. In other embodiments, the reactivesurface is a surface that includes a reducing reagent. Using systems ofthe invention, metal clusters of any type may be produced.

Different combinations of solvent and voltage allow the generation ofmetal cluster ion species with different oxidation states individuallyor collectively, including those that are labile towarddisproportionation when stored in bulk. The chemical environment of thedeposition medium is independently manipulated and this allows for thetransformation of metal cluster ion species. The different chemicalenvironment of the target deposition medium allows, for example,synthesis of clusters; synthesis of organometallic clusters includingcatalysts; and metal tagging by reaction with other neutral molecules.

Accordingly, the invention provides direct synthesis of small chargedmetal clusters and ligated metal clusters from metal salts without theuse of chemical reducing agents. Systems and methods of the inventionoffer an alternative to laser and plasma based methods of generatingmetal and other molecular clusters. The systems and methods of theinvention allow for the generation of charged metal clusters atatmospheric pressure under mild conditions. The charged metal clustersmay be directly deposited into liquids and onto surfaces at atmosphericpressure to cause chemical reactions or to prepare surfaces, includingcatalytically active surfaces.

Systems and methods of the invention eliminate the need for chemicalreducing agents to produce metal clusters, and the need for cappingligands to stabilize the metal clusters. Systems and methods of theinvention eliminate the need for lasers and vacuum chambers for theproduction of ligated and unligated charged metal clusters. Rather,clusters are formed at atmospheric pressure under mild heatingconditions and can be ligated by ion/molecule or solution phasereactions. The bare metal clusters are building blocks, which allowligands, especially weaker one, to be attached.

Systems and methods of the invention provide a continuous atmosphericpressure metal cluster ion source at a low cost. The source can behandled and transported with ease. The source can be controlled to allowa specific amount of deposition onto a surface or into a liquid similarto the vacuum based experiments. Surface patterning of metal clusterscan be done at atmospheric pressure in addition to the traditionalmethod of in vacuum. Prepared surfaces can be catalytically active.

Reactions in ambient environment or vacuum can exploit the uniquechemical properties of small molecular clusters. This allows in-situgeneration of catalysts in a reactor.

Functionalized Substrates

In other embodiments, the invention provides substrates for surfaceenhanced Raman Spectroscopy and methods of making thereof. In certainembodiments, the invention provides a substrate for surface enhancedRaman Spectroscopy that includes a substrate and at least one discretespot on the substrate that is modified for surface enhanced RamanSpectroscopy. The spot includes an aggregate of uncapped metalnanoparticles, in which each nanoparticle of the aggregate maintains itsindividual features. Other embodiments provide a functionalizedsubstrate including a substrate, and a plurality of discrete structuredfeatures on the substrate, each structured feature including anaggregate of uncapped metal nanoparticles, in which each nanoparticle ofthe aggregate maintains its individual features.

The substrate may include a plurality of discrete spots. Typically,although not required, the spots are patterned onto the substrate. Thesubstrate may additionally include a sample. The sample may be below theaggregate of uncapped metal nanoparticle. Alternatively, the sample maybe above the aggregate of uncapped metal nanoparticle. The substrate maybe composed of a variety of different types of materials. In anexemplary embodiment, the substrate includes a metal. The uncapped metalnanoparticles may include any type of metal, such as a novel metal. Incertain embodiments, the metal is silver. In certain embodiments, theuncapped metal nanoparticles are uniform in size. Typically, theaggregates will be non-spherically shaped.

Other embodiments provide methods for producing a functionalizedsubstrate. Such methods involve providing a substrate, and spraying,under ambient conditions, metal ions from a droplet emitter onto adiscrete location on the substrate, thereby producing an aggregate ofmetal nanoparticles at the discrete location on the substrate. Themethods may additionally involve moving to at least one other discretelocation on the substrate, and spraying, under ambient conditions, metalions from a droplet emitter onto the other discrete location on thesubstrate, thereby producing an aggregate of uncapped metalnanoparticles at the other discrete location on the substrate. Themethod may be repeated a plurality of times to produce an array ofdiscrete spots. Methods of the invention may additionally involvedepositing a sample on the substrate. The sample may be deposited priorto the spraying step. Alternatively, the sample may be deposited afterthe spraying step.

Other embodiments provide methods for producing a functionalizedsubstrate that involve providing a substrate and a focusing mask, andspraying, under ambient conditions, metal ions from a droplet emittertoward the focusing mask such that the ions interact with the mask andare focused to a discrete location on the substrate, thereby producingan aggregate of metal nanoparticles at the discrete location on thesubstrate. In certain embodiments, the mask is a conductive mask. Inother embodiments, the mask is a non-conductive mask. In certainembodiments, the mask may selectively block a portion of the metal ions.For example, the mask may selectively block positive metal ions.Alternatively, the mask may selectively block negative metal ions. Incertain embodiments, moving the mask generates another aggregate ofmetal nanoparticles at another discrete location on the substrate.Methods of the invention may additionally involve depositing a sample onthe substrate. The sample may be deposited prior to the spraying step.Alternatively, the sample may be deposited after the spraying step.

Silver is a widely used SERS material (Asiala et al., Analyst 2011, 136,4472-4479) and the plasmon resonance of silver nanostructures are knownto be tunable throughout the visible to mid-infrared regions of theelectromagnetic spectrum (Jensen et al., J Phys Chem B 2000, 104,10549-10556). Metal electrolytic spray ionization (described in greaterdetail in U.S. patent application Ser. Nos. 61/877,528 and 61/880,219,the content of each of which is incorporated by reference herein itsentirety) spots on top of desired locations, both on top of previouslydeposited analyte and prior to analyte deposition. (FIG. 16, panel A).Both the NP-on-top and the NP-below configurations showed uniformlydistributed silver NP's in SEM images. (FIG. 16, panel B). The sizes andshapes of the particles were polydispersed, yet the polydispersedmorphology was uniform across each spot. (FIG. 19 panels A-C). On theone hand, the polydispersity gave the surface a wide range of surfaceplasmon resonance, making the spot SERS active when using lasers ofdifferent wavelengths [532, 633 and 785 nm, FIG. 20 using crystal violetand Rhodamine 6G FIG. 21 as probing molecules]. On the other hand, theuniform distribution created numerous evenly distributed hot spots. Therobustness of this SERS surface is greatly enhanced by these features.Both the NP-on-top and the NP-below surfaces showed similar SERSenhancements, FIG. 16 panel B.

A single metal electrode spray ionization emitter generates typical ioncurrent of at least 10 nA. Silver(Ag⁺) containing ions dominates as canbe observed by an atmospheric pressure interface to a mass spectrometer(Li et al., Angew. Chem., Int. Ed. 2014, 53, 3147-3150). The diameter ofthe emitter tip was typically 1-5 μm. The spray plume started with this˜μm scale diameter and after travelling toward landing surface inambient air along the electrical gradient for ˜5 mm, the plume diameterhad expanded to 3-5 mm. Mapped by an Ion CCD (Hadjar et al., J. Am. Soc.Mass Spectrom. 2011, 22, 612-623), the charge distribution of the plumemaximized at its center and dropped towards the edges, very slowly inthe beginning and then more rapidly (FIG. 22). This “pseudo uniformity”in the central region was evident when examining the prepared structuresby optical and electron microscopy. The coverage of the deposited silverions can be controlled by regulating the deposition time and estimatingthe deposition current and spot size. Better coverage values werecalculated from the accurately measured spot sizes and the loggeddeposition currents (Li et al., Angew. Chem., Int. Ed. 2014, 53,3147-3150). The deposited metal ions created circles of nanoparticleswhich were used in SERS analysis by Raman microscopes.

Using copper foil as the support material, a coverage dependent studywas carried out to investigate its effect on SERS enhancement. As shownin FIG. 23, the Raman signal for crystal violet (1 μM in MeOH, 2 μLdropcast over the ˜3 mm AgNP spot) increased more than 10 times when thesilver coverage increased from 1.6 ML to 5.5 ML and continued toincrease until the CCD detector was saturated at 9.9 ML. SEM images ofthese surfaces are shown in FIG. 17.

When the surface coverage was low (1-3 ML), only single nano particlesand small numbers of aggregates were observed. As the silver coverageincreased through further deposition, the granules/particles started togrow larger. At 5.5 ML coalescence and aggregation of neighboringparticles was observed. As more silver was deposited (7.0 ML),aggregation gradually became universal making it difficult to identifyindividual particles. This coverage controlled and surface anchored insitu nanostructure fabrication produced uncapped nanoparticles with farbetter uniformity than those typical of solution-phase experiments ((Liet al., Angew. Chem., Int. Ed. 2014, 53, 3147-3150), in which sizedistribution can only be controlled by capping agents. An importantphenomenon is that the individual NP features were maintained duringthis aggregation process creating numerous 1-5 nm gaps and crevicesacross the surface. This is likely due to the fact that the particleswere anchored to the metal surfaces during their growth. These types ofnano junctions and nano gaps are believed to be ideal for creating SERShot spots (Asiala, Z. D. Schultz, Analyst 2011, 136, 4472-4479; andFang, N. H. Seong, D. D. Dlott, Science 2008, 321, 388-392). When thecoverage was further increased 9.9 ML, these aggregates grew even biggerwhile keeping their non-spherical shapes. The aggregated nature of theprepared NP structures was also evident from measurements by dark filledhyper-spectral imaging (HSI) in which the scattering spectra showedmultiple colors of photons scattered from the aggregated nanoparticlesgenerated on top of an ITO coated glass slide. The SERS peak intensitysummarized in Tables 3 and 4 in the Examples and the enhancement factorswere calculated using a previous reported method (Greeneltch et al.,Anal. Chem. 2013, 85, 2297-2303; and Chakraborty et al., Journal ofPhysical Chemistry Letters 2013, 4, 2769-2773), and also described inthe Examples below.

If it is assumed that all current measured is that of singly chargedmetal ions with a circular landing spot of 3 mm diameter, 10 nA currentis equivalent to 0.03 ML/minute. It would take 5 hr to prepare a 10 MLspot of this size. A higher landing current density can be achieved bypositioning the emitter closer to the surface or by increasing the sprayvoltage from 1.5 kV to say ˜2.5 kV. However, these operations alsoincreased the fluctuation of both landing current and spot size, makingit difficult to estimate coverage accurately. By placing a mask made ofnon-conductive material (or electrically floated conductive material)placed on top of the deposition surface the landing current densitycould be increased reproducibly. The local electric field that enabledthe increase in current density is believed to be generated through aself-charging mechanism by charge buildup on the non-conducting materialduring the ion deposition experiment) The simplest form of this idea isrealized by applying a perforated plastic foil on top of the depositiontarget. In typical experiments, this increased landing current densityby a factor of ˜9 even though a 5-25% decrease in total ion current wasobserved. This procedure also enabled a more uniform currentdistribution across the deposition region. Arrays of AgNP containingspots were created in a single deposition experiment, simply by usingmasks with an array of apertures (FIGS. 18 and 24-26).

Besides silver coverage, support materials were also found to have greatinfluence on SERS activity of the deposited silver nanostructures, as isthe case for other modification methods (Greeneltch et al., Anal. Chem.2013, 85, 2297-2303; Asiala et al., Analyst 2011, 136, 4472-4479; andMurty et al., Langmuir 1998, 14, 5446-5456). Generally, the flatpolished slide supports gave much lower SERS enhancement under the sameconditions (10 ML Ag coverage, Table 2 in Examples below). Copper,aluminum and gold foils gave the highest averaged SERS enhancementfactors exceeding 108 and reaching 1010. While brass, stainless steeland silver foils gave very weak enhancements. Although detailedmechanisms are not clear, gold foil support demonstrates thatdisplacement plating (Lu et al., J. Am. Chem. Soc. 2007, 129, 1733-1742;and Liu et al., J. Am. Chem. Soc. 2013, 135, 11752-11755) (even ifpossible when depositing silver ions onto other metals with loweroxidizing potential) is not critical in the generation of SERS activesurfaces by this method. The flexible foils used here serve as anefficient SERS sampling medium allowing drop casting, wiping, spincoating and spray deposition of samples. More importantly, samples canbe present on the surface first and then hot spots can be generated insitu by depositing silver ions onto the sample spot (NP-on-top).Enhanced Raman signals were observed for all these methods. The SERSsurfaces stayed active after ambient storage (1 Month) and afterexposing to electron SEM analysis.

The metal electrolytic spray ionization deposition nanostructurefabrication method is a ‘green’, one-pot, ambient preparation methodthat eliminates vacuum, laser, and solution procedures associated withconventional nanofabrication. Micron scale patterns can easily be madefor SERS imaging. For each SERS substrate, only sub nanogram amounts ofsilver are consumed. Operation under atmospheric pressure furtherreduces the cost and increases the ease of nanoscale surfacemodification. Metal electrolytic spray ionization deposition may servewell as a ready alternative to sputtering or vapor deposition in certainapplications. Beyond SERS applications described in this report, metalelectrolytic spray ionization deposition should find applications inrelated fields such as plasmonic superstructures and catalysis.

Collection of Ions without or after Mass-Selective Analysis

Systems and methods for collecting ions that have been analyzed by amass spectrometer are shown in Cooks, (U.S. Pat. No. 7,361,311), thecontent of which is incorporated by reference herein in its entirety. Inanother embodiment, ions may be collected in the ambient environment(ambient pressure but still under vacuum) without mass analysis (SeeExamples herein). The collected ions may then be subsequently analyzedby any suitable technique, such as infrared spectrometry or massspectrometry.

Generally, the preparation of microchips arrays of metal ions firstinvolves the ionization of the metal. The metal ions can be produced byany of the methods discussed above. The ions can then be focused andcollected using methods described below or can first be separated basedon their mass/charge ratio or their mobility or both their mass/chargeratio and mobility. For example, the ions can be accumulated in an ionstorage device such as a quadrupole ion trap (Paul trap, including thevariants known as the cylindrical ion trap and the linear ion trap) oran ion cyclotron resonance (ICR) trap. Either within this device orusing a separate mass analyzer (such as a quadrupole mass filter ormagnetic sector or time of flight), the stored ions are separated basedon mass/charge ratios. Additional separation might be based on mobilityusing ion drift devices or the two processes can be integrated. Theseparated ions are then deposited on a microchip or substrate atindividual spots or locations in accordance with their mass/charge ratioor their mobility to form a microarray.

Whether or not mass-selection is used, the microchip or substrate ismoved or scanned in the x-y directions and stopped at each spot locationfor a predetermined time to permit the deposit of a sufficient number ofmolecules to form a spot having a predetermined density. Alternatively,the gas phase ions can be directed electronically or magnetically todifferent spots on the surface of a stationary chip or substrate. Themolecules are preferably deposited on the surface with preservation oftheir structure, that is, they are soft-landed.

In embodiments in which ions are collected without prior separation, thecollection surface is operably coupled to receive the spray includingthe ions, as illustrated in FIG. 1. In embodiments that first usemass-selection, the surface is located behind the detector assembly ofthe mass spectrometer. In embodiments that use an ion focusing device,the surface for ion landing is located after the ion focusing device.

In embodiments that use mass-selection prior to ion landing, the highvoltages on the conversion dynode and the multiplier are turned on andthe ions are detected to allow the overall spectral qualities,signal-to-noise ratio and mass resolution over the full mass range to beexamined. In the ion-landing mode, the voltages on the conversion dynodeand the multiplier are turned off and the ions are allowed to passthrough the hole in the detection assembly to reach the landing surfaceof the plate (such as a gold plate). The surface is grounded and thepotential difference between the source and the surface is 0 volts.

An exemplary substrate for soft landing is a gold substrate (20 mm×50mm, International Wafer Service). This substrate may consist of a Siwafer with 5 nm chromium adhesion layer and 200 nm of polycrystallinevapor deposited gold. Before it is used for ion landing, the substrateis cleaned with a mixture of H₂SO₄ and H₂O₂ in a ratio of 2:1, washedthoroughly with deionized water and absolute ethanol, and then dried at150° C. A Teflon mask, 24 mm×71 mm with a hole of 8 mm diameter in thecenter, is used to cover the gold surface so that only a circular areawith a diameter of 8 mm on the gold surface is exposed to the ion beamfor ion soft-landing of each mass-selected ion beam. The Teflon mask isalso cleaned with 1:1 MeOH:H₂O (v/v) and dried at elevated temperaturebefore use. The surface and the mask are fixed on a holder and theexposed surface area is aligned with the center of the ion optical axis.

Any period of time may be used for landing of the ions. Inmass-selection embodiments, between each ion-landing, the instrument isvented, the Teflon mask is moved to expose a fresh surface area, and thesurface holder is relocated to align the target area with the ionoptical axis. After soft-landing, the Teflon mask is removed from thesurface.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

Various modifications of the invention and many further embodimentsthereof, in addition to those shown and described herein, will becomeapparent to those skilled in the art from the full contents of thisdocument, including references to the scientific and patent literaturecited herein. The subject matter herein contains important information,exemplification and guidance that can be adapted to the practice of thisinvention in its various embodiments and equivalents thereof.

EXAMPLES Example 1: Catalytic Activity of Nanoparticles Generated byMetal Spray Deposition

A sample reaction was used to probe the catalytic reactivity of goldnanoparticles prepared as described above. UV-Vis absorption was used tomonitor the reaction progression under different conditions (FIG. 5). Anonline in situ catalyst generation experiment was also carried outwithout the use of capping ligands, which are prerequisites inconventional solution-phase nanoparticle synthesis. Data analysisindicates that the catalytic activity (per Au atom) of the AuNP preparedthis way is several orders of magnitude higher that those preparedoffline (FIG. 6). The catalytic activity is surface-area related andthis indicates the presence of even smaller (<100 atoms) particles inthe in situ catalysis method by directly spraying Au⁺ to the reactionliquid (FIG. 6).

Example 2: Preparation of Nanoparticle Alloy

The electrospray method was used to introduce different noble metals ona surface or into a solution in a controllable way. A “one-pot”nanoparticle alloy preparation was demonstrated as one application ofthis capability. Compared to multiple steps involved in bulk synthesis,simply switching the spraying nozzle is a much easier way of introducingdifferent metal sources to the particle cores. FIG. 7 is a transmissionelectron microscope (TEM) image of a nanoparticle composed of a silvercore surrounded by and attached to smaller gold spheres. FIG. 8 is a TEMimage of a nanoparticle composed of a gold core surrounded by silversphere. Silver has lower electron density results in low color tone inthe TEM figure.

Example 3: Generating Catalysts for Other Reactions (ATRPPolymerization)

The Cu(I)-ligand species (FIG. 9) are actually key structures forcatalysis of Atom Transfer Radical Polymerization (ATRP). These specieswere collected similarly to the procedure used above in amounts (˜1.5mg) much less than those used in the literature. When used as a catalystin the synthesis of polystyrene, polymers in the amount of gram wereobtained (FIG. 10).

Example 4: Silver Metal Cluster Ions

Systems of the invention were used to produce silver metal clusters.FIG. 14A is an ion chronogram of Ag₃ ⁺ (m/z 320-328) as a function oftemperature. FIG. 14B is a representative mass spectrum of 1 mM silveracetate that has been passed through a heated coiled loop at 215° C. The[Ag₃]⁺ represents 17% of the total ion current.

Example 5: Silver Metal Cluster Ions in Alcohol Solvent

Systems of the invention were used to produce silver metal clusters thatwere directed to a vessel including isopropyl alcohol. The silver metalclusters reacted with the isopropyl alcohol. FIG. 15 panel A is a massspectrum showing atmospheric ion/molecule reaction of silver clusterswith isopropyl alcohol. Ligation of 1 and 2 isopropyl alcohol wereobserved with the silver trimer. FIG. 15 panel B shows MS² of m/z 384confirming the ligation of 1 isopropyl alcohol. FIG. 15 panel C showsMS² of m/z 443 and FIG. 15 panel D shows MS³ of m/z 384 confirming theligation of two metal ligands.

Example 6: Functionalized Substrates and Methods of Making andPatterning Substrates

The example relates to functionalized substrates, such as substratesfunctionalized for surface enhanced Raman Spectroscopy, methods ofmaking thereof, and methods of patterning surfaces.

Metallic nanoparticles have attractive properties in catalysis,photonics, and chemical sensing (Eustis et al., Chem. Rev. 2005, 105,1547-1562; and Jain et al., Acc. Chem. Res. 2008, 41, 1578-1586). Ramanspectroscopy is a powerful non-destructive technique (Biggs et al.,Chem. Rev. 1999, 99, 2957) the sensitivity of which can be significantlyimproved through Surface enhanced or and tip enhanced Raman Scattering(SERS, TERS) (Jeanmaire et al., Journal of Electroanalytical Chemistryand Interfacial Electrochemistry 1977, 84, 1-20; Wang et al., Analyst2013, 138, 3150-3157; and Stockle et al., Chem. Phys. Lett. 2000, 318,131-136). The enhancement arises due to an analytes' proximity tointense localized fields created by nano tips, nanoparticles, or nanoparticle assemblies (Hao et al., J. Chem. Phys. 2004, 120, 357-366;Genov et al., Nano Lett. 2004, 4, 153-158; and Negri et al., Chem.Commun. 2014, 50, 2707-2710). The capability to modify, coat and patternsurfaces with nano structures is important, not only for SERS, but alsofor material functionalization (Nge et al., Journal of MaterialsChemistry C 2013, 1, 5235-5243), in situ analysis (Li et al., Nature2010, 464, 392-395), as well as large scale nano material fabrication(Wang et al., Anal. Chem. 2014). Conventional modified surfaces areconstructed by delivering intact nanoparticles to the target locationsvia drop casting or spin coating (Xia et al., Journal of Vacuum Science& Technology A 2013, 31; Osberg et al., Adv. Mater. 2012, 24, 6065;Cyriac et al., Analyst 2012, 137, 1363-1369). However, the difficulty inpositioning discrete particles with control over orientation, position,and degree of aggregation, means that drop casting of nanoparticles hasnot been widely used in high throughput SERS analysis. Immobilized andshell isolated nano systems (Huang et al., Nature 2010, 464, 392-395;and Greeneltch et al., Anal. Chem. 2013, 85, 2297-2303) do address theseissues but tedious vacuum preparation procedures significantly increasethe cost of such approaches.

Ion/surface collisions including or ion soft-landing has been used infabricating surface structures under vacuum (Rauschenbach et al., Phys.Chem. C 2012, 116, 24977-24986; Lei et al., Science 2010, 328, 224-228;and Cyriac et al., Chem. Rev. 2012, 112, 5356-5411). Recently a metalelectrolytic spray ionization deposition method (Li et al., Angew.Chem., Int. Ed. 2014, 53, 3147-3150) has been developed that is capableof generating noble metal ions directly from solid under ambientconditions as precursors for nanoparticle synthesis.

The example provides methods for in situ fabrication of SERS activespots and patterns by metal electrolytic spray ionization deposition todesired locations where they automatically yield assemblies ofnanostructure. Metal electrolytic spray ionization deposition was usedfor surface fabrication of 2D patterned nano structures forsurface-enhanced Raman spectroscopy (SERS). Silver nanoparticle (AgNP)containing spots were created in desired locations accurately usingmasks with appropriate patterns of apertures and with focusing of theion spray by charge buildup on the edges of the apertures. No cappingagent was used and the morphology and SERS activity of the NP structureswere controlled by degrees of coverage of deposited ions. The NPstructures were created either as sampling media, or directly on top ofsample containing regions. The evenly distributed hot spots had ahighest average enhancement factor of 5×10⁸. The surfaces are SERSactive using lasers of different wavelengths (532, 633, 785 nm).

Materials and Chemicals

The support surfaces used in this experiment included ITO slides,aluminum coated (˜100 nm) microscope glass slides (Deposition ResearchLab, St. Charles, Mo.), gold (120 nm) with Titanium (100 nm) adhesionlayer coated microscope glass slide (Deposition Research Lab, St.Charles, Mo.), ITO coated glass slides (1.1 mm thickness, 1″×3″,(Nanocs, New York, N.Y.)), heavy duty aluminum foil of 0.01 mm thickness(Durable Packaging International, Wheeling, Ill.), soft annealed copperfoil of 0.05 mm thickness (McMaster-Carr, Elmhurst, Ill.). Silver foil,Stainless Steel foils, and gold foil of 0.01 mm thickness were purchasedfrom Aldrich Chemical Company (Milwaukee, Wis.). P400 silicon carbideabrasive paper (Buehler, Ill.) were used to polish oxide layer androughen surfaces when needed. TEM grids (Electron Microscopy Science)were used as received.

The metal electrodes used for MESID were assembled as previouslydescribed.^([22]) HPLC grade acetonitrile, methanol (Chromasolv,Sigma-Aldrich) was used in related experiments. Crystal violet,Rhodamine 6G (reagent grade, Sigma-Aldrich), was used as received.

Chemical Instrumentation

An ambient ionizing and deposition set up was constructed and used toaccurately log the amount of ions delivered onto any collecting surface(Li et al., Angew. Chem., Int. Ed. 2014, 53, 3147-3150). Briefly, awire-in nanoESI source were loaded with anhydrous acetonitrile subjectedto a high voltage of ˜1.5 kV. The ionic species generated by the ionsource were checked by an Orbitrap mass spectrometer (LTQ Orbitrap XL,Thermo, CA) before and after deposition. Metal containing ions weredirected to a grounded target surface. The recombination current throughthe ground was monitored and logged once every second. Target surfaceswere grounded and positioned 5-10 mm away from the tip of the MESIemitter, static and under ambient condition. Monolayer coverage (ML) wascalculated based on the total deposited charge and the size of spot. Thesizes of the deposition spot was measured afterwards using electron oroptical microscopes. Perforated masks was used when focused ion beam orspecific spot sizes were needed. The ML was used to as a measure for thedegree of surface modification. The spatial distribution of ion currentat the deposition surface was measured using an IonCCD detector system(OI Analytical, College Station, Tex., USA), similar as previouslydescribed (Baird et al., Int. J. Mass Spectrom. 330, 277, 2012. TheIonCCD is a pixelated charge detector consisting of an array of 21 μmwide TiN pads or pixels 1.5 mm in height, separated by 3 μm. When ionscome in contact with the floated electrode surface they are neutralizedand their charge is stored over a user determined integration time.Following the integration time the charge on each pixel is read outserially and the resulting signal is reported in the form of a digitalnumber (dN). The detector array and associated electronics are housed ina stainless steel enclosure with a 1.5 mm wide, 49 mm long slit exposingthe detector surface. A detailed description of the detector operationis provided by Hadjar et al. (J. Am. Soc. Mass Spectrom. 22, 612, 2011).Unless otherwise noted, integration time was set to 100 ms and 25 V wasapplied to the stainless steel housing of the detector.

An optical microscope (Olympus BX-51) equipped was used to obtain brightfield, dark field and fluorescence image in various spectrum regions.The simultaneous single particle dark field imaging and spectra wasmeasured using a hyperspectral assembly (Cytoviva HSI) comprising anOlympus BX-41 microscope fitted with a Dage high resolution camera andSpecim V10E spectrometer (Bootharaju et al., Rsc Advances 2, 10048,2012). The characteristic scanning range is from 400-1000 nm, dividedinto 462 bands which gives a spectral resolution of +/−1.5 nm. Therelative scattering intensity of the particle is a determinant of itscolor with 640, 550 and 460 nm, assigned to red, green and bluerespectively.

SEM images and EDAX data were taken on a FEI Philips XL-40 ScanningElectron Microscope with a Schottky field emission gun. High resolutionTEM images of the samples were obtained using a JEOL 3010 instrumentwith a UHR pole piece. Specimens for TEM analysis were prepared byplacing lacey carbon grid on top of the collecting surface. SeveralRaman instruments equipped with different lasers were used to evaluateprepared SERS active surfaces. The first one was an Alpha-SNOM 300 Sconfocal Raman microscope (WITec GmbH, Germany) with a 532 nm laser asexcitation source. The large area scan was done on an area of 4 mm×4 mmwith 200 spots per line. Large area optical image was taken using imagestitch option in the software equipped with this Raman instrument. Thesecond was an Alpha 300 confocal Raman microscope (WITec GmbH, Germany)with a 633 nm laser as excitation source. The third one was a nearInfrared Raman imaging microscope (Olympus BX60) with a 785 nm laser.Raman signals were collected using objective lenses, laser powerintensities and with an integration time denoted individually in eachfigure.

All the Raman spectra shown here is background corrected. The backgroundcorrection was done using the WITec instrument provided software,initially the spectrum was fitted with a best fit polynomial and thenthat was subtracted from the original spectra. Raman images weregenerated based on the intensity of Raman peaks using the WITecsoftware.

Enhancement Factors Calculation, Uniformity Evaluation and OtherConsiderations

The enhancement factor (EF) was calculated based on the measured Ramanspectra. At first the SERS intensities were compared with normal Ramanintensities, corrected for the number of molecules under the laser spot.The formula to measure the EF is given as (Stiles et al., Annu. Rev.Anal. Chem. 1, 601, 2008):

${EF} = \frac{I_{SERS}\text{/}N_{surface}}{I_{normal}\text{/}N_{bulk}}$

I_(SERS), I_(normal), N_(bulk), N_(surface) are observed SERSintensities of arising from monolayer coating of analyte molecule (here,crystal violet (CV) or Rhodamine 6G (R6G)) on the Ag nanoparticle spot,Raman intensity of analyte molecule in absence of nanoparticle (normalRaman signal), number of analyte molecules excited under laser spot forbulk specimen and number of analyte molecules under the laser spot on Agnanoparticles, respectively. I_(SERS) and I_(normal) were taken from thenormalized (for power and acquisition time) intensity of C-H in planebending mode (Raman shift at 1176 cm-1 for CV and 1365 cm⁻¹ for R6G).N_(bulk) and N_(surface) values are computed by using the formula givenbelow.

N _(surface)=4πr ² ·C·A·N

where r, C, A, N are average particle radius of the Ag nanoparticles ofthe spot, surface density of the analyte monolayer, area of the laserspot and average number of particles per square micrometer area,respectively. The average particle radius r was taken (from SEMmeasurement) as 32 nm, surface density of analyte molecule C calculatedas 105/μm², area of laser spot (50× objective, Numerical Aperture=0.55)diameter was 3 μm (A=7.1 μm²), number of particles per square micrometerN is (from SEM measurement) 255.

N _(bulk) =N _(A) ·A·h·ρ/M;

A is area of the laser spot, h is penetration depth of the laser, ρ isdensity of the solid analyte (0.83 g/cm³ in case of crystal violet),molecular weight of the analyte (in this work, 408 for crystal violetand 479 for Rhodamine 6G). The laser spot was 3 μm diameter; penetrationdepth of laser h was taken as 20 μm.

Using these parameters and the previously quoted equation, the highestEF for the AgNP structured copper foil was calculated to be 3.8×10¹⁰,Table 2 below. Al, Au foils, was calculated to be 6.9×10⁶ and 2.2×10⁷.

TABLE 2 Average Signal Intensity and Enhancement Factor for AgNP ondifferent materials Support Material Highest Peak Intensity (1176 cm⁻¹)EF Copper Foil 12921 3.86E+07 Gold foil 7310 2.18E+07 Aluminum Foil 23026.88E+06 Copper Tape 3731 1.11E+07 Brass foil 40 1.19E+05 StainlessSteel Foil 181 5.41E+05 Silver foil n.a. n.a. ITO coated slide 308.96E+04 Aluminum coated slide 864 2.58E+06 Penny coin AgNP first 5901.76E+06 Penny coin Sample first 471 1.41E+06 in situ deposition on 3139.35E+05 sample

Other Considerations on the Enhancement Factor: Resonance Contributionand Laser Induced Damage

Molecular electronic resonance Raman (RR) and surface-enhanced Ramaneffects was observed to increase Raman signal synergistically. Crystalviolet has a wide absorption spectrum with an absorbance maximum rangingfrom 420 to 600 nm depending on the environment pH. The resonance Ramancontribution to enhancement factor (EFRR) can be as much as 10^(5˜7)when the laser wavelengths meet the electronic excitation energy of theanalytes (Dieringer et al., J. Am. Chem. Soc. 131, 849, 2009); andKleinman et al., J. Am. Chem. Soc. 133, 4115, 2011). This might be onereason for the extremely high signal intensity when C.V. sample wereprobed under the 532 laser, as showing in the following figure. This maybe a contributing factor that complicates the obtained SERS enhancementfactor.

The 785 nm laser is far from the resonance of crystal violet. Theseexperiment combinations shall give enhancement factors withoutinterference of resonance contribution. Shown in FIG. 19, panel C, nearIR laser gave average enhancement factor of 2×10⁵. This decrease,however, may also be due to the different interaction between creatednanostructures with the near IR laser. For this reason, R6G was testedunder 633 nm (which is far from R6G's resonance) for better comparison.The result is shown in the below FIG. 20 as well as summarized in Table3 below.

Another interesting phenomenon observed this SERS experiment is that thehigh SERS signal always experienced a decrease during recording of thespectrum. If the sample was slightly moved, the signal would jump(sometime beyond the CCD saturation level) to a high value and thenimmediately decrease within the 1 second integration time. In theimaging mode, Raman spectra over each pixel were taken with 0.01 secondintegration time and then the sample stage moved to next pixel. Muchhigher signal intensities, as well as enhancement factors was obtainedin this mode.

Showing in FIG. 20, a highest signal found in the is integration scansis only 9 times higher than a highest signal in 0.01 s integrationscans. This could be the result of thermal desorption of the moleculefrom the hotspot driven by laser, or the result of laser induced meltingof nanoparticles since no capping agent was used to protect the AgNPs.Even this 0.01 integration time gave a lot better results, most of theEF reported in Table 1 are still based on 1 second integration time withthe consideration that most Raman spectrometers are built without imagescanning function.

In summary, Resonance contribution may have brought up the overallenhancement factor but laser induced damage could have brought down theactual enhancement factor. Future modification on the surface may giveeven better performance for SERS applications.

SERS Uniformity of the Modified Surfaces

For real applications of SERS, the surface uniformity is an importantmeasure that determines the robustness of the experiment. Densely andevenly distributed hotspots would be ideal for rapid Raman analysis. Theuniformity of the modified surface were evaluated by repeatingmeasurements on randomly selected regions in the same spot. Thecorresponding values for the AgNP structures built on three differentsupport materials were summarized in Table 3.

TABLE 3 SERS intensity of band 11 cm⁻¹ in different regions of thespots, 10 ML Ag coverage, 8.6 mW, 633 nm laser excitation Support %Material region 1 region 2 region 3 region 4 region 5 mean RSD Copper9747 12733 11510 12921 11204 11623 11% Foil Al foil 2302 1213 2173 16901739 1823 24% Au foil 7310 5324 4880 6160 5259 5787 17%

For AgNP arrays composed of small spots, uniformity evaluation was doneby Raman imaging of the different areas composing the spot. As shown inthe below FIG. 21, these spots are effectively identical for SERSpurposes.

In sum, the net enhancement signal is very uniform. Considering thevariance of analyte's surface distribution brought by dropcasting, theSERS uniformity and reproducibility of the prepared surface isexceptional.

SEM Images of Ag⁺ Modified Surfaces

A series of surfaces were tested as supported materials for the in situpreparation of AgNP by metal electrolytic spray ionization deposition.Different SERS performance were found for the different supportmaterials as summarized in Table 2. These modified surfaces showdifferent morphologies as imaged by scanning electron microscope. Evenamong the “good” substrates, different morphologies can be observed. SeeFIGS. 22 and 27-28.

The Deposition Plume: Spatial Flux Distribution, Coverage, Morphologyand SERS Signal

Once loaded with anhydrous acetonitrile and being in contact with highvoltage, the metal electrolysis spray ionization (MESI) source readilygenerated silver containing ions as dominating ion signal observed by anatmospheric pressure sampling mass analyzer, as discussed previously (Liet al., Angew. Chem., Int. Ed. 2014, 53, 3147-3150). The diameter of thecharged droplet emitter tips were typically 1-5 μm. After progression inambient air along the electric gradient for ˜5 mm, the spray plume'sdiameter expanded to 1-5 mm. The metal ions' distribution in thisexpanded plume may result in an uneven distribution of precursor ionconcentration on the collecting surfaces. Mapped by an Ion CCD (FIG. 29panels A-B), for a spray plume of ˜3 mm diameter, the current maximizein the center and dropped slowly less than 30% for the first 1.5 mmoutward from the center. For the next 1 mm, the current dropped a lotmore rapidly and accounted for the rest 70%. For this reason, it wasassumed a uniform distribution of deposited metal ions for most areasinside the deposition circle. This “uniform in the center” assumption islargely valid as observed by optical and electron microscopes. (FIG. 29panels B and D and FIG. 21) Monolayer coverage was controlled bydeposition time with an estimation based on deposition current and spotsize. The actual monolayer coverage of each experiment was calculatedafterwards with the accurately measured spot sizes and the depositioncurrents logged by a computerized system (Li et al., Angew. Chem., Int.Ed. 2014, 53, 3147-3150).

Hyperspectral Imaging of Prepared Nanoparticles and Aggregates

Surface plasmon resonance (SPR) Dark filed hyper spectra imaging (HSI)scattering spectrum was carried out to analyze one sample of aggregatednanoparticle prepared by depositing ˜10 ML silver ions (FIGS. 30-31).

Ion Beam Focusing and Creating Surface Patterns Using Metal ElectrolyticSpray Ionization Deposition with Masks

Static patterns of nanoparticle containing spots was created by puttingmasks between the ion emitter and the deposition targets. Groundedconductive mask (FIGS. 24-26) creates a negative pattern by simplyblocking ions depositing to the positive regions. Non-conductive andfloated conductive masks, however, provides additional focusing effectthat gives higher flux and smaller (than the mask holes' dimensions)spots.

In a particular investigation, ion optical simulations were performedusing the ray tracing program SIMION to test the expectation thatdirecting a spray of ions towards a small aperture in a non-conductingsurface would build up change on the surface as a result of ions landingthere and that this potential would act to focus the ions that movethrough the aperture. A charge of 1000 v was assumed and the initial ionenergy was a few tens of volts. The results (FIGS. 32A-B) show that thefinal spot size was 100 times smaller than the aperture diameter,indicating very strong focusing.

1-20. (canceled)
 21. A method for producing a functionalized substrate,the method comprising: providing a substrate; providing a focusing mask;and spraying, under ambient conditions, metal ions from a dropletemitter toward the focusing mask such that the ions interact with thefocusing mask and are focused to a discrete location on the substrate,thereby producing an aggregate of metal nanoparticles at the discretelocation on the substrate.
 22. The method according to claim 21, furthercomprising: moving to at least one other discrete location on thesubstrate; and spraying, under ambient conditions, metal ions from adroplet emitter toward another location on the focusing mask such thatthe ions interact with the other location on the focusing mask and arefocused to another discrete location on the substrate, thereby producingan aggregate of uncapped metal nanoparticles at the other discretelocation on the substrate.
 23. The method according to claim 21, whereinthe method is repeated a plurality of times to produce an array ofdiscrete spots.
 24. The method according to claim 22, further comprisingdepositing a sample on the substrate.
 25. The method according to claim24, wherein the sample is deposited prior to the spraying step.
 26. Themethod according to claim 24, wherein the sample is deposited after tothe spraying step.
 27. The method according to claim 21, wherein thesubstrate comprises a metal.
 28. The method according to claim 21,wherein the uncapped metal nanoparticles are uniform in size.
 29. Themethod according to claim 21, wherein the focusing mask is anon-conductive focusing mask.
 30. The method according to claim 21,wherein the focusing mask is a conductive focusing mask.
 31. A methodfor producing a functionalized substrate, the method comprising:providing a substrate; providing a focusing mask; and spraying, underambient conditions, metal ions from a droplet emitter toward thefocusing mask such that the ions interact with the focusing mask and arefocused to a discrete location on the substrate, wherein the focusingmask may selectively block a portion of the metal ions, therebyproducing an aggregate of metal nanoparticles at the discrete locationon the substrate.
 32. The method according to claim 31, furthercomprising: moving to at least one other discrete location on thesubstrate; and spraying, under ambient conditions, metal ions from adroplet emitter toward another location on the focusing mask such thatthe ions interact with the other location on the focusing mask and arefocused to another discrete location on the substrate, thereby producingan aggregate of uncapped metal nanoparticles at the other discretelocation on the substrate.
 33. The method according to claim 31, whereinthe method is repeated a plurality of times to produce an array ofdiscrete spots.
 34. The method according to claim 32, further comprisingdepositing a sample on the substrate.
 35. The method according to claim34, wherein the sample is deposited prior to the spraying step.
 36. Themethod according to claim 34, wherein the sample is deposited after tothe spraying step.
 37. The method according to claim 31, wherein thesubstrate comprises a metal.
 38. The method according to claim 31,wherein the uncapped metal nanoparticles are uniform in size.
 39. Themethod according to claim 31, wherein the focusing mask is anon-conductive focusing mask.
 40. The method according to claim 31,wherein the focusing mask is a conductive focusing mask.